The present invention relates to the production of novel synthetic antigens based upon information derived from DNA and/or protein sequences and to the use of those antigens in the production of vaccines, diagnostic reagents, and the like. More specifically, this invention relates to synthetic antigenic polypeptides, which when used alone, as a polymer or upon coupling to a carrier, immunologically correspond to a T cell and B cell determinant portion of hepatitis B virus surface antigen (HBsAg).
Viral hepatitis continues to rank as one of the most important unconquered diseases of mankind. The general term, viral hepatitis, refers principally to hepatitis A (infectious hepatitis) and to hepatitis B (serum hepatitis), although other known viruses such as yellow fever virus, Epstein-Barr virus and cytomegalovirus can cause hepatitis in man. Hepatitis is particularly known for its focal attack on the liver (Greek, hepar), but the disease also influences other organs.
In 1965, Blumberg discovered an antigen circulating in the blood of certain human beings [J. Am. Med. Assoc., 191, 541 (1965) and Ann. Int. Med., 66, 924 (1967)]. This substance was subsequently found by Prince to be the surface antigen of hepatitis B virus (HBsAg) that is produced in abundance by individuals who are chronically infected with the agent [Proc. Natl. Acad. Sci. (USA), 60, 814 (1968)].
HBsAg has been the subject of extensive immunochemical characterization. Serologic studies show that several strains of the hepatitis B virus (HBV) have one or more determinants in common, which is designated a. Each strain also has two other determinants: either d or y and either w or r. Thus, there are four possible types of the virus: adw, ayw, adr and ayr. The specificity of HBsAg is associated with a single polypeptide [Gold et al., J. Immunol., 117, 1404 (1976) and Shih et al., J. Immunol., 120, 520 (1978)], the entire 226 amino acid sequence of which is established from the nucleotide sequence of the S gene [Tiollais et al., Science, 213, 406 (1981)] of HBV [Valenzuela et al., Nature (London), 280, 815 (1979); Galibert et al., Nature (London), 281, 646 (1979) and Pasek et al., Nature (London), 282, 575 (1979)].
There is an urgent need for a hepatitis B vaccine for groups which are at an increased risk of acquiring this infection. These groups include health care and laboratory personnel, and individuals requiring (1) maintenance hemodialysis; (2) repeated blood transfusions or the administration of blood products; (3) treatment with immunosuppressive or cytotoxic drugs and (4) treatment for malignant diseases and disorders associated with depression of the immune response. In addition, a vaccine is needed for individuals living in certain tropical areas where hepatitis B infection is prevalent.
Hepatitis A and B viruses, however, do not multiply significantly in cell culture, and there is no current source of laboratory propagated virus for vaccine preparation. Indeed, there has been a repeated failure to transmit hepatitis B virus (HBV) serially in tissue or organ cultures which has hampered progress towards the development of a conventional vaccine [Zuckerman, Amer. J. Med. Sci., 270, 205 (1975)].
Classically, a vaccine is manufactured by introducing a killed or attenuated organism into the host along with suitable adjuvants to initiate the normal immune response to the organism while, desirably, avoiding the pathogenic effects of the organism in the host. That approach suffers from several well known limitations. These vaccines are complex and include not only the antigenic determinant of interest but many related and unrelated deleterious materials, any number of which may, in some or all individuals, induce an undesirable reaction in the host.
For example, vaccines produced in the classical way may include competing antigens which are detrimental to the desired immune response, antigens which include unrelated immune responses, nucleic acids from the organism or culture, endotoxins and constituents of unknown composition and source. These vaccines, generated from complex materials, inherently have a relatively high probability of inducing competing responses even from the antigen of interest.
In the past, antigens have been obtained by several methods including derivation from natural materials, coupling of a hapten to a carrier and by recombinant DNA technology. Sela et al. [Proc. Nat. Acad. Sci. (USA), 68, 1450 (1971); Science, 166, 1365 (1969); and Adv. Immun., 5, 129 (1966)] have also described certain synthetic antigens.
Certain "synthetic" antigens have been prepared by coupling small molecules (for example, dinitrophenol) to carriers (such as bovine serum albumin), thus producing antigens which cause the production of antibody to the coupled small molecule. The carrier molecule is often necessary because the small molecule itself may not be "recognized" by the immune system of the animal into which it is injected. This technique has also been employed in isolated instances to prepare antigens by coupling polypeptide fragments of known proteins to carriers, as described in the above-referenced Sela et al articles.
While this hapten-carrier technique has served the research community well in its investigations of the nature of the immune response, it has not been of significant use in the production of antigens which would play a role in diagnostic or therapeutic modalities. One reason for that deficiency is that to select and construct a useful antigenic determinant from a pathogen (e.g., hepatitis B virus) by this technique, one must determine the entire protein sequence of the pathogen to have a reasonable chance of success. Because of the difficulty of this task, it has rarely, if ever, been done.
Recombinant DNA technology has opened new approaches to vaccine technology and has the advantage that the manufacture begins with a monospecific gene; however, much of this advantage is lost in actual production of antigen in E. coli, or other organisms. In this procedure, the gene material is introduced into a plasmid which is then introduced into E. coli which produces the desired protein, along with other products of the metabolism, all in mixture with the nutrient. This approach is complicated by the uncertainty as to whether the desired protein will be expressed in the transformed E. coli.
Moreover, even though the desired protein may be produced, there is uncertainty as to whether or not the protein can be harvested or whether it will be destroyed in the process of E. coli growth. It is well known, for example, that foreign or altered proteins are digested by E. coli. Even if the protein is present in sufficient quantities to be of interest, it must still be separated from all of the other products of the E. coli metabolism, including such deleterious substances as undesired proteins, endotoxins, nucleic acids, genes and unknown or unpredictable substances.
Finally, even if it were possible (or becomes possible through advanced, though necessarily very expensive, techniques) to separate the desired protein from all other products of E. coli metabolism, the vaccine still comprises an entire protein which may include undesirable antigenic determinants, some of which are known to initiate adverse responses. Indeed, it is known that certain proteins which could otherwise be considered as vaccines include an antigenic determinant which induces serious cross reference or side reactions that prevent the use of the material as a vaccine.
It is also possible, using hybridoma technology, to produce antibodies to viral gene products. Basically, hybridoma technology allows one to begin with a complex mixture of antigens and to produce monospecific antibodies later in the process. In contrast, the present invention is the reverse process, in that we start with a relatively high purity antigenic determinant and thus avoid the necessity for purification of the desired antigenic product.
Hybridoma antibodies are known to exhibit low avidity and low binding constants, and therefore, have limited value. Moreover, in hybridoma technology, one must rely on the production of the antibody by cells which are malignant, with all of the attendant concerns regarding separation techniques, purity and safety.
Hybridoma production relies upon tissue culture or introduction into mice, with the obvious result that production is costly and there is an inherent variability from lot to lot.
In addition, it is difficult to make hybridomas that secrete antibodies to molecules which comprise only a small percentage of the complex mixture with which one must start, or which are poorly immunogenic and are overshadowed by stronger, dominant antigens.
Previous studies by Arnon et al., Proc. Nat. Acad. Sci. (USA), 68, 1450 (1971), Atassi, Immunochemistry, 12, 423 (1975) and Vyas et al., Science, 178, 1300 (1972) have been interpreted by those authors to indicate that short linear amino acid sequences are, in general, unlikely to elicit antibodies reactive with the native protein structure. It was thought that for most regions of most molecules, antigenic determinants resulted from amino acid residues well separated in the linear sequence but close together in the folded protein. The exact three dimensional conformation of the polypeptides used to elicit antibodies was thought to be critical in most cases, even for those antigens involving amino acids close together in a sequence.
For example, Sela thought it necessary to synthesize a rather elaborate loop structure to elicit an anti-lysozyme response. Atassi engineered many elaborate molecules, each intended to mimic the tertiary structure of the target protein. And Vyas concluded that the three dimensional conformation of hepatitis B surface antigen was a critical factor in eliciting antibodies reactive with that native structure.
Sutcliffe et al., Nature, 287, 801 (1980) discovered that antibodies to linear polypeptides react with native molecules, and recent investigations have shown that relatively short chemically synthesized polypeptides can elicit antibodies reactive with almost any region of an exposed surface of a protein [Green et al., Cell, 28, 477 (1982)]. Moreover, since amino-acid sequences can now be determined rapidly with nucleic acid sequencing technology, synthetic polypeptides can be synthesized to make vaccines of a precision not previously possible. Thus, elaborate biosyntheses are unnecessary, uneconomical and obsolete.
U.S. Pat. No. 4,415,491 to Vyas discloses a series of peptides that correspond to the a determinant of hepatitis B virus surface antigen. Although no data is presented concerning the protection of a host, the peptides are described as being useful in a hepatitis vaccine preparation.
Current vaccines for HBV consist of subviral components of the virus surface coat (HBsAg) purified from the plasma of chronically HBV-infected donors and inactivated [McAuliffe et al., Rev. Infect. Dis., 2, 470 (1980)]. Clinical trials have demonstrated the safety and efficacy of current HBsAg vaccines but such vaccines are limited in supply and are relatively expensive, particularly for those countries with the highest incidence of HBV disease. Chemically synthesized polypeptides, therefore, offer considerable advantages in terms of cost and safety of HBV vaccination programs.
It is known that antisera to synthetic polypeptides predicted from the nucleotide sequence of various regions of the S gene of HBV react with native HBsAg by radioimmunoprecipitation [Lerner et al., Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981)] and commercial solid-phase radioimmunoassays for anti-HBsAg [Gerin et al., in Viral Hepatitis, Szmuness et al (eds.), 49-55 (1982)].
It has been recently determined that a pathogen-related protein can be immunologically mimicked by the production of a synthetic polypeptide whose sequence corresponds to that of a determinant domain of the pathogen-related protein. Such findings are reported by Sutcliffe et al., Nature, 287, 801 (1980) and Lerner et al., Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981).
Moreover, Gerin et al., Proc. Natl. Acad. Sci. (USA), 80, 2365 (1983) have recently shown limited protection from hepatitis B virus upon immunization with carrier bound-synthetic polypeptides having amino acid sequences that correspond to the amino acid sequence of a determinant portion of HBsAg; in particular, residues 110-137.
The construction of a synthetic HBsAg vaccine, however, may require in addition to synthetic polypeptides corresponding to B cell (antibody-producing) epitopes, synthetic polypeptides corresponding to non-overlapping T cell determinants.
By way of further background, three cellular components of the immune system are B cells (bursaor bone marrow-derived lymphocytes), T cells (thymus-derived lymphocytes) and macrophages. B cells circulate in the blood and the lymph fluid and are involved in the production of antibodies. T cells amplify or suppress the response by B cells.
Macrophages, on the other hand, are involved in presenting and concentrating antigens to B and T cells. Moreover, macrophages secrete several biologically active mediators that regulate the type and magnitude of both T and B cell responses either by enhancing or suppressing cell division or differentiation. Macrophages are nonspecific and react against any foreign antigen. T and B cell, however, are antigen-specific and react via cell membrane receptors that are specific for the particular antigen.
In mice, the in vivo antibody production to HBsAg is regulated by at least 2 immune response (Ir) genes, one in the I-A subregion (Ir-HBs-1) and one in the I-C subregion (Ir-HBs-2) of the murine H-2 complex. It is observed that immunization with a chemically synthesized peptide corresponding to the d determinant did not distinguish between high and non-responder murine strains. Milich et al., J. Immunol., 130, 1401 (1983). This suggests that Ir-restriction may occur through T cell recognition of additional, perhaps nonoverlapping, regions of the molecule.
The linkage between major histocompatibility complex and the regulation of immune responsiveness to HBsAg in mice has been extended to the human immune response by the report of an association between HLA-DR phenotype and nonresponsiveness to a recent trial HBsAg vaccine. Thus, the construction of synthetic HBsAg vaccine may require, in addition to B cell epitopes, a sufficient diversity of T cell determinants to accommodate the genetic variation in epitope recognition of an outbred human population.
The following information would be very valuable in developing a synthetic HBsAg vaccine: (1) whether synthetic peptide fragments representing a highly restricted region of the native HBsAg (i.e., about 6 amino acids) can induce a T cell proliferative response, which, as with native HBsAg, is regulated by H-2 linked genes; (2) whether T cell recognition sites overlap with antibody binding sites; (3) whether multiple T cell recognition sites exist on HBsAg and if so whether the site(s) recognized depend on the H-2 genotype of the responding strain; (4) whether the T cell site(s) recognized determine the specificity and quality of the humoral response; and (5) whether human HBsAg-primed T cells are activated by the same determinants that induce T cell proliferation in mice.